ProT2® MHC Class II Tetramers

Figure 1: ProT2® MHC Class II Tetramer

Detect and separate single antigen-specific CD4+ T cells with our new best in class ProT2® MHC Class II Tetramers

ProT2® MHC Class II Tetramers allow you to detect single antigen-specific CD4+T cells accurately by flow cytometry. They can also be used to separate cells for culture, expansion and further study. ProT2® Tetramers enable you to study in depth CD4+ T cell immune responses in a wide range of disease areas including autoimmune disease, cancer and infectious diseases. ProT2® Tetramers are made by ProImmune’s experienced team who have been providing superior MHC reagents for more than 15 years, including our world-leading Pro5® MHC Class I Pentamers. Custom ProT2® Tetramers are available within approximately 6 weeks from ordering.

ProT2® staining for CD4+ T cell epitopes from CMV

Data from Prof Paul Moss group, University of Birmingham, UK

Figure 2. PBMCs from CMV-positive and CMV-negative donors were stained with DRB1*07:01/PDDYSNTHSTRYVTV ProT2® Tetramer. Plots on the left show cell samples from a CMV-positive donor; plots on the right show cell samples from a CMV-negative donor. A population of CD4+ tetramer-positive cells is visible in the upper right quadrant of the plot from CMV-positive donor cells stained with ProT2® Tetramer. All plots were derived by gating on live, CD19-negative, CD14-negative, CD3-positive lymphocytes.

Traditional functional assays for studying antigen-specific CD4+ T cells, such as proliferation or cytokine secretion, are not truly antigen-specific. The direct detection of antigen-specific CD4+ T cells has been difficult, due to the low frequency of these T cells (down to 1 in 100,000 white blood cells) and the low functional avidity, caused by low MHC/peptide-TCR affinity, reduced TCR clustering and coreceptor effects. The optimized manufacturing process for ProT2® Tetramers helps to address these issues by improving reproducibility and sensitivity of CD4+ T cell detection.

Available Quantities

Fluorescent Label

ProT2® MHC Class II Tetramers are delivered with a choice of standard R-PE or APC labels. Alternatively customers can purchase and conjugate our ProM2® MHC Class II Monomers to any other suitable avidin or streptavidin reagent.

Catalog ProT2® MHC Class II Tetramers

A broad range of MHC Class II complexes specific for a wide range of infectious diseases such as Influenza, CMV, hepatitis B and C, HIV, EBV, as well as cancer-related epitopes are available as ProT2® Tetramers. Catalog ProM2® Tetramers are despatched in 1-2 days for Stock items, and 3 weeks for Available items.

Custom ProT2® MHC Class II Tetramers

Custom ProT2® Tetramers for your allele/peptide of interest are available within approximately 6 weeks from ordering. Peptide sequences are subject to prior evaluation by ProImmune for binding. Once a peptide sequence is accepted for custom synthesis we will synthesize both the peptide and MHC tetramer reagent.

ProT2® MHC Class II Tetramer staining

ProImmune has extensive experience in testing ProT2® MHC Class II Tetramers in-house in a variety of applications. This enables us to support our customers fully with their relevant application requirements, helping them to make best use of our technology.

ProT2® staining for CD4+ T cell epitope from Influenza

Data from ProImmune

Figure 3. PBMCs from healthy donors were stimulated with peptide PKYVKQNTLKLAT (Influenza A HA 307­­­‑319), cultured for 13 days, and stained with DRB1*01:01/PKYVKQNTLKLAT ProT2® Tetramer. Plots on the left shows a cell sample that has not been stimulated with peptide; plots on the right show the same cells that have been stimulated with peptide. A population of CD4+ tetramer-positive cells is visible in the upper right quadrant of the plot from peptide-stimulated HLA-DRB1*01:01 donor cells. All plots were derived by gating on live, CD19-negative lymphocytes.

ProT2® MHC Class II Tetramers may be used to study CD4+ T cell immune responses in infectious diseases such as CMV (see figure 1 above) and HCV, and in vaccine studies.

ProT2® staining for CD4+ T cell epitopes from HCV

Figure 4. PBMCs from HCV-positive donors were stained with ProT2® Tetramer. The left plot shows a population of CD4+ tetramer-positive cells in the upper right quadrant from HCV-positive HLA-DRB1*04:01 donor cells stained with DRB1*04:01/SGIQYLAGLSTLPGNPAIASL ProT2® Tetramer. The right plot shows a population of CD4+ tetramer-positive cells in the upper right quadrant from HCV-positive HLA-DRB1*01:01 donor cells stained with DRB1*01:01/TLLFNILGGWVAA ProT2® Tetramer. All plots were derived by gating on live, CD19-negative, CD14-negative, CD3-positive lymphocytes.

ProT2® staining for CD4+ T cell epitopes from HCV

Data from Prof Ellie Barnes group, University of Oxford, UK

Figure 5. PBMCs from post HCV-vaccinated donors were stimulated with peptide NFPYLVAYQATVCARA and cultured. Cells were stained on day 12 and day 22 with DRB1*15:01/NFPYLVAYQATVCARA ProT2® Tetramer. A population of CD4+ tetramer-positive cells is visible in the upper right quadrant of the plots from HLA-DRB1*15:01 donor cells. All plots were derived by gating on live, CD3-positive lymphocytes.

To aid detection of low frequency populations that are beyond the limit of detection of standard direct Class II tetramer staining, expansion [1, 2] or enrichment with magnetic beads [3, 4] may be required; although some very low affinity antigens may not give detectable staining.